Our studies are directed toward establishing whether the current concepts of steroid hormone action involving free diffusion from blood into cells and binding to receptors are correct. Studies of hyperestrogenic postmenopausal women with endometrial cancer revealed that they have a 2-\to 3-fold increase in plasma-free estradiol as compared to hypoestrogenic women with osteoporosis. The absolute concentration of free estradiol is about 10-12 M, which is far below the reported dissociation constant of the estrogen receptor (KD = 10-9 - 10-12 M). Since these values are probably too high because of errors associated with current methods of receptor analysis based upon bound hormone, we have developed a new approach involving measurement of free ligand. We are also comparing steroid binding in serum and cells from primates including new world monkeys that have extraordinarily high circulating levels of glucocorticoids, estrogens, and androgens. The results of these studies suggest that a steroid bound to the specific blood transport proteins (CBG and SHBG) may be the biologically important fraction. (D)